Metabolic Capacity Differentiates Plenodomus lingam from P. biglobosus Subclade ‘brassicae’, the Causal Agents of Phoma Leaf Spotting and Stem Canker of Oilseed Rape (Brassica napus) in Agricultural Ecosystems

In contrast to the long-lasting taxonomic classification of Plenodomus lingam and P. biglobosus as one species, formerly termed Leptosphaeria maculans, both species form separate monophyletic groups, comprising sub-classes, differing considerably with epidemiology towards Brassicaceae plants. Considering the great differences between P. lingam and P. biglobosus, we hypothesized their metabolic capacities vary to a great extent. The experiment was done using the FF microplates (Biolog Inc., Hayward, CA, USA) containing 95 carbon sources and tetrazolium dye. The fungi P. lingam and P. biglobosus subclade ‘brassicae’ (3 isolates per group) were cultured on PDA medium for 6 weeks at 20 °C and then fungal spores were used as inoculum of microplates. The test was carried out in triplicate. We have demonstrated that substrate richness, calculated as the number of utilized substrates (measured at λ490 nm), and the number of substrates allowing effective growth of the isolates (λ750 nm), showed significant differences among tested species. The most efficient isolate of P. lingam utilized 36 carbon sources, whereas P. biglobosus utilized 60 substrates. Among them, 25–29 carbon sources for P. lingam and 34–48 substrates for P. biglobosus were efficiently used, allowing their growth. Cluster analysis based on Senath criteria divided P. biglobosus into two groups and P. lingam isolates formed one group (33% similarity). We deduce the similarities between the tested species help them coexist on the same host plant and the differences greatly contribute to their different lifestyles, with P. biglobosus being less specialized and P. lingam coevolving more strictly with the host plant.


Introduction
Two fungal species belonging to the genus Plenodomus (formerly Leptosphaeria) coexist in Brassicaceae plants, share similar life patterns and cause the disease called stem canker or blackleg [1][2][3]. For many years the isolates were classified as one species Leptosphaeria maculans (Desm.) Ces et de Not., but numerous researchers postulated their separation. Already in 1927, based on morphological characteristics and growth rate observed on agar media, Cunningham [4] described two forms of L. maculans. Some isolates were growing slowly, and the aerial mycelium was sparse, whereas the sporulation of the other isolates was less abundant, but the mycelium grew fast and the brownish pigment was excreted extracellularly. The finding did not lead to the description of the new species and it was forgotten until the widespread cultivation of oilseed rape (Brassica napus L.).
After brassica crop plants became more common, the symptoms of the stem canker started to appear more often, and L. maculans-the causal agent of the disease started to be the seedling stage, but mostly on the first few leaves, depending on the time of 'ascospore showers' [29]. This, in turn, greatly depends on the cultural practices of the farmers [30] but primarily on weather conditions [31]. The large dataset of spore counts from air samples, studied in relation to the weather parameters over 17 years in the central-west part of Poland, has shown that increased average air temperature and rainfall shifted the detection of the first spores by 22 days and the day of the maximum spores by 50 days, which resulted in much earlier occurrence of disease symptoms and their higher impact on yield [32].
Considerable yield losses due to stem canker occur worldwide every year [11,31,33,34]. Usually, the annual yield loss reported in oilseed rape is 10%, but it may reach 50% [31], or even all plants may get infected in particular years or regions. In some areas of intensive oilseed rape cultivation in the world, such as the one located in China, the main losses are caused by P. biglobosus sub-clade 'brassicae' [25] and recently also by the sub-clade 'canadensis' [26], and not by P. lingam [35]. Scenarios describing the fast expansion of the stem canker into oilseed rape growing areas in North and Central China along the Yangtze River showed the great potential threat to the stability of oilseed rape production if the crop was attacked by P. lingam in the absence of resistant cultivars [36]. New breeding programs have been launched in search of traits with the potential to mitigate outbreaks of stem canker. Based on genome-wide association studies, the sources of resistance against P. lingam have been identified in Chinese and Canadian spring and winter oilseed rape cultivars [37,38].
Nowadays, the diversification of the species is not only based on morphological traits but it is supported by genetic characteristics, starting from the size and sequence of the ITS region [39], minisatellites [40], and (in P. lingam) also the presence of several avirulence genes [41,42]. In P. lingam the race composition of fungus populations greatly depends on the specific resistance genes present in oilseed rape [43]. In contrast, by now no resistance to P. biglobosus has been found. Plant infection by P. biglobosus leads to premature ripening and decreased plant yield [34,44]. On the other hand, it is presumed that the fungus occupies the same niche as P. lingam and, being less virulent, it contributes to stem canker control. The studies on P. lingam and P. biglobosus became a model for the investigations of genetic relationships between the pathogen and the host plant [39].
Both Plenodomus species are hemibiotrophic, which means they first develop in live plant organs and then subsequently colonize the senescing plants [45,46]. The colonization of identical plant tissues at the same time would require similar metabolic capacities. Aware of great differences of genotypic and phenotypic characteristics between P. lingam and P. biglobosus, we hypothesized their metabolic capacities also vary. The aim of this study was to check this hypothesis by comparing the metabolic capacities of P. lingam and P. biglobosus sub-clade 'brassicae'.

Results
The metabolic capacities of Plenodomus isolates were tested using the FF microplates (Biolog) including 95 carbon sources. The values of substrate richness (R) calculated as the number of utilized substrates (A490 nm) and the number of substrates allowing effective growth of fungal isolates (A750 nm), demonstrated significant differences among tested species of Plenodomus. The pathogens were able to utilize from 36 (P. lingam-PL1) to 60 (P. biglobosus-PB1) of the tested carbon sources. Among them, from 25 to 29 carbon sources for P. lingam and from 34 to 48 substrates for P. biglobosus were efficiently used, allowing the growth of the fungi.
The average R values of utilized substrates for P. lingam and P. biglobosus were 40 and 57, respectively, as well as allowing for effective fungal growth were 28 and 41, respectively. The tested strains were not able to utilize and grow on all 95 carbon sources. The rates in the average well color development (AWCD) and average well-density development (AWDD) indices values were used to identify the differences in the response of tested fungal isolates on various consumption of carbon sources and growth response, respectively. The results indicated that both AWCD and AWDD values were significantly higher for all tested strains of P. biglobosus than P. lingam. This tendency was observed through most of the incubation period ( Figure 1).
spectively. The tested strains were not able to utilize and grow on all 95 carbon sources. The rates in the average well color development (AWCD) and average well-density development (AWDD) indices values were used to identify the differences in the response of tested fungal isolates on various consumption of carbon sources and growth response, respectively. The results indicated that both AWCD and AWDD values were significantly higher for all tested strains of P. biglobosus than P. lingam. This tendency was observed through most of the incubation period ( Figure 1).
The carbon assimilation profiles and growth intensity profiles of tested pathogens were obtained by analyses of substrate guilds and are summarized in Figure 2. The carbon assimilation profiles and growth intensity profiles of tested pathogens were obtained by analyses of substrate guilds and are summarized in Figure 2. Higher differences were observed for substrate utilization connected with fungal respiration and mitochondrial activity than that noted for growth response. Qualitative analysis of the proportions between the particular guilds of utilized substrates indicated that P. biglobosus was capable of metabolizing a defined group of compounds in almost equal proportions by all tested isolates (PB1, PB2, PB3), while substrate utilization and growth intensity responses of P. lingam varied little between tested isolates (PL1, PL2, PL3). P. biglobosus strains exhibited significantly higher metabolic preferences than P. lingam for amino acids, carboxylic acids, and miscellaneous categories of C-substrates ( Figure 3). Higher differences were observed for substrate utilization connected with fungal respiration and mitochondrial activity than that noted for growth response. Qualitative analysis of the proportions between the particular guilds of utilized substrates indicated that P. biglobosus was capable of metabolizing a defined group of compounds in almost equal proportions by all tested isolates (PB1, PB2, PB3), while substrate utilization and growth intensity responses of P. lingam varied little between tested isolates (PL1, PL2, PL3). P. biglobosus strains exhibited significantly higher metabolic preferences than P. lingam for amino acids, carboxylic acids, and miscellaneous categories of C-substrates ( Figure 3).
Plenodomus lingam preferentially utilized various carbon sources and polymers, while amides/amines were degraded at a very low level by all tested isolates belonging to both species of Plenodomus. Significant differences in growth responses of the two tested fungal pathogen species on various carbon sources guilds were observed only for amino acids, carboxylic acids, and polymers ( Figure 4). Generally, amino acids and carboxylic acids were used the most by P. biglobosus, while polymers were preferred by P. lingam.
To reveal the differences in metabolic capacity and growth intensity of tested strains during incubation time, the average color development and density development in the wells of FF Biolog plates were calculated for each day of incubation through 10 days (240 h). The results showed significant diversity in fungal metabolism and growth intensity between the compared species of Plenodomus. It should be noted that during 240 h of fungal incubation, the highest catabolic activity of P. biglobosus was observed after 144 h of incubation, whereas the use of the substrates by P. lingam was the highest after 240 h of incubation. Moreover, all tested strains of P. lingam exhibited significantly lower metabolic potential and growth than P. biglobosus. Both fungal species were better characterized by metabolic capacity (carbon source utilization) than growth rate ( Figure 2).  Plenodomus lingam preferentially utilized various carbon sources and polymers, while amides/amines were degraded at a very low level by all tested isolates belonging to both species of Plenodomus. Significant differences in growth responses of the two tested fungal pathogen species on various carbon sources guilds were observed only for amino acids, carboxylic acids, and polymers ( Figure 4). Generally, amino acids and carboxylic acids were used the most by P. biglobosus, while polymers were preferred by P. lingam.  To reveal the differences in metabolic capacity and growth intensity of tested strains during incubation time, the average color development and density development in the wells of FF Biolog plates were calculated for each day of incubation through 10 days (240 h). The results showed significant diversity in fungal metabolism and growth intensity between the compared species of Plenodomus. It should be noted that during 240 h of fungal incubation, the highest catabolic activity of P. biglobosus was observed after 144 h of incubation, whereas the use of the substrates by P. lingam was the highest after 240 h of  The results of metabolic potential obtained in this study allowed the grouping of the tested strains into two major clusters ( Figure 5).
between the compared species of Plenodomus. It should be noted that during 240 h of fungal incubation, the highest catabolic activity of P. biglobosus was observed after 144 h of incubation, whereas the use of the substrates by P. lingam was the highest after 240 h of incubation. Moreover, all tested strains of P. lingam exhibited significantly lower metabolic potential and growth than P. biglobosus. Both fungal species were better characterized by metabolic capacity (carbon source utilization) than growth rate ( Figure 2).
The results of metabolic potential obtained in this study allowed the grouping of the tested strains into two major clusters ( Figure 5). Strains classified as P. biglobosus constituted group A with higher metabolic capacity. The cluster denoted with B comprised strains belonging to P. lingam displaying weaker abilities to metabolize the carbon sources compared to L. biglobosa. The strains of P. lingam showing good growth on tested carbon sources clustered together, while L. biglobosa created two sub-clusters. This clustering was connected with the results of the metabolic profile of all tested substrates and growth intensity profile on various carbon sources. The patterns of carbon source utilization are presented in Figure 6.
Additionally, the ratio between mitochondrial respiration (OD490 nm) and fungal growth (OD750 nm) calculated for the different groups of substrates showed the diverse metabolic efficiency of the fungal isolates (Figure 7).  Substrates belonging to amino acids and carboxylic acids were the most stressful for both tested species showing lower metabolic efficiency on these organic sources. However, carbohydrates were more stressful for P. lingam than for P. biglobosus fungal strains. What is more, a stressful metabolic situation, indicated by the ratio of both AWCD to AWDD (Figure 8) was met using especially arbutin, L-glutamin acid and L-threonine by P. lingam and glucose-1-phosphate, salicin, bromosuccinic acid, Substrates belonging to amino acids and carboxylic acids were the most stressful for both tested species showing lower metabolic efficiency on these organic sources. However, carbohydrates were more stressful for P. lingam than for P. biglobosus fungal strains. What is more, a stressful metabolic situation, indicated by the ratio of both AWCD to AWDD (Figure 8) was met using especially arbutin, L-glutamin acid and L-threonine by P. lingam and glucose-1-phosphate, salicin, bromosuccinic acid, ɣ-hydroxy butyric acid, L-malic acid, quinic acid, D-saccharic acid, L-alanyl glycine, L-aspartic acid, L-glutamic acid, L-serine and putrescine.

Discussion
The results of this study confirmed the working hypothesis about the differences in metabolic capacities between P. lingam and P. biglobosus sub-clade 'brassicae'. Despite considerable variation in metabolic activities of individual isolates, both species formed two separate groups with distinct metabolic potential. According to the Sneath criteria, the FF MicroPlate C-sources utilization profiles, as well as the growth intensity of the analyzed strains, was distinct for P. lingam and P. biglobosus. The utilization calculated based on absorbance value at 490 nm divided the isolates of P. biglobosus into two clusters (with 66% similarity of the substrate utilization profiles) and one cluster for P. lingam -hydroxy butyric acid, L-malic acid, quinic acid, D-saccharic acid, L-alanyl glycine, L-aspartic acid, L-glutamic acid, L-serine and putrescine. Substrates belonging to amino acids and carboxylic acids were the most stressful for both tested species showing lower metabolic efficiency on these organic sources. However, carbohydrates were more stressful for P. lingam than for P. biglobosus fungal strains. What is more, a stressful metabolic situation, indicated by the ratio of both AWCD to AWDD (Figure 8) was met using especially arbutin, L-glutamin acid and L-threonine by P. lingam and glucose-1-phosphate, salicin, bromosuccinic acid, ɣ-hydroxy butyric acid, L-malic acid, quinic acid, D-saccharic acid, L-alanyl glycine, L-aspartic acid, L-glutamic acid, L-serine and putrescine.

Discussion
The results of this study confirmed the working hypothesis about the differences in metabolic capacities between P. lingam and P. biglobosus sub-clade 'brassicae'. Despite considerable variation in metabolic activities of individual isolates, both species formed two separate groups with distinct metabolic potential. According to the Sneath criteria, the FF MicroPlate C-sources utilization profiles, as well as the growth intensity of the analyzed strains, was distinct for P. lingam and P. biglobosus. The utilization calculated based on absorbance value at 490 nm divided the isolates of P. biglobosus into two clusters (with 66% similarity of the substrate utilization profiles) and one cluster for P. lingam (33% similarity). Moreover, the increase of fungal biomass calculated based on absorbance value at 750 nm showed identical results with the more stringent similarity (33%). This means that despite having identical host plants and life cycles [47], the species P.

Discussion
The results of this study confirmed the working hypothesis about the differences in metabolic capacities between P. lingam and P. biglobosus sub-clade 'brassicae'. Despite considerable variation in metabolic activities of individual isolates, both species formed two separate groups with distinct metabolic potential. According to the Sneath criteria, the FF MicroPlate C-sources utilization profiles, as well as the growth intensity of the analyzed strains, was distinct for P. lingam and P. biglobosus. The utilization calculated based on absorbance value at 490 nm divided the isolates of P. biglobosus into two clusters (with 66% similarity of the substrate utilization profiles) and one cluster for P. lingam (33% similarity). Moreover, the increase of fungal biomass calculated based on absorbance value at 750 nm showed identical results with the more stringent similarity (33%). This means that despite having identical host plants and life cycles [47], the species P. lingam and P. biglobosus maintain separate metabolic capacities. The differences partially explain why P. lingam and P. biglobosus manifest different ways of inhabiting oilseed rape plants, resulting in contrasting effects on plant health and yield. Based on our experiment, the species P. biglobosus uses numerous and variable sources of carbon and under natural conditions, it grows very quickly [34,44], whereas P. lingam is restricted to a narrower range of C-containing nutrients, which is the most likely reason for its slower growth. The response of both studied species to carbohydrates shows no statistical differences, but they tend to be in favor of P. biglobosus species. Moreover, P. biglobosus grows significantly better on carboxylic acids and amino acids.
Warm and humid weather increases the reproduction potential of P. biglobosus [48] and leads to fast colonization of oilseed rape stems [49], leading to severe stem canker symptoms [50]. High metabolic capacities help P. biglobosus species achieve quite spectacular success in inoculum production, reaching millions of pycnidiospores, that are able to colonize adjacent plants [51,52]. Moreover, the fungus increases its reproduction capacities by up to a few thousand of ascospores per cubic meter of the air [31,53], which incredibly increases the ecological success of this species.
To a great extent spore germination and mycelial growth [54] and hence, also the stem canker disease is controlled by plant resistance and fungicide application [55], especially when the latter are well-timed by the decision support systems [29,56]. However, naturally created mechanisms such as fast growth, abundant reproduction, and higher levels of resistance to commonly used fungicides are in favor of the survival of P. biglobosus. It was usually found as the first species on oilseed rape [1,33,37,46], and in some countries, out of the two species, only P. biglobosus has been reported on oilseed rape by now [57].
In contrast, P. lingam grows slowly, but we have demonstrated that it utilizes substrates more efficiently than P. biglobosus. Moreover, the number of substrates causing substrate stress is much lower as compared to P. biglobosus. Considering the ability to produce toxins contributing to the pathogenesis, this species achieved the ecological success in a different way than P. biglobosus.
Field populations of P. lingam display a high evolutionary potential and can overcome major resistance genes within a few years [58]. The most dramatic example was the break of resistance introduced to the Australian cultivar Surpass from B. rapa subsp. sylvestris, which was broken within one year [59]. Many other examples show the population of P. lingam may shift rapidly in response to the common use of oilseed rape cultivars carrying single resistance (Rlm) genes [60,61]. These genes exert strong selection pressure on corresponding avirulence effector genes of P. lingam [42].
The evolution of the pathogen toward virulence [62] resulted in a vast search for stable quantitative resistance (QR) loci to stem canker [63]. It was proved that QR resistance increases the durability of qualitative resistance of oilseed rape to P. lingam [64,65]. Recently, numerous significant quantitative trait loci (QTL) for QR were detected on several chromosomes belonging to A or C genomes, of which eight were repeatedly detected across diverse environments of oilseed rape cultivation, located in Australia, France, and the United Kingdom [66]. Association mapping confirmed the high number of genomic regions involved in oilseed rape QR to stem canker [67]. These stable QTLs can be used for enhancing QR in elite germplasm via marker-assisted or genomic selection strategies [66]. Additionally, comparative mapping pinpointed several R genes coding for nucleotide-binding leucinerich repeat (LRR) receptors, which helps to combine QR and specific resistance to P. lingam, conferred by R genes. Similar to the other plant diseases, the control of P. lingam relying on resistant varieties is challenging and must be based on QR resistance as well as efficient working and diversified Rlm genes of various origins and their careful and well-planned deployment [58].
Great progress in resistance breeding over the last three decades has helped to control stem canker. The breakdown of resistance to stem canker reported in Australia has been averted in commercial cultivars of oilseed rape [68]. However, durable resistance based on several QTLs may bring questions about the fitness cost [69]. Decreased yield frequently encountered in resistant cultivars is not acceptable to farmers. The recent introduction of pro-ecological programs, such as the European Green Deal, with a toxic-free environment and zero pollution of water, air, and soil, will promote the further pursuit to utilize diseaseresistant cultivars of crop plants. There is a hope the use of super parasites and natural products will reduce the pressure exerted on P. lingam and it will reduce its further spread and virulence towards Brassicaceae plants. An increased investigation of P. biglobosus influence on P. lingam is needed. Its results may help to exploit natural competition between these species in occupying oilseed rape plants. This, in turn, requires detailed studies of plant tissues composition to compare metabolic capacities of P. lingam and P. biglobosus with the availability of substrates in various plant organs, stages of plant development, and differences between the commercially used cultivars of oilseed rape. Ideally, the investigations should compare the results of metabolic capacities of all subclades within P. lingam and P. biglobosus, with a high number of isolates representing each of these taxonomic groups.

Fungal Strains
The studies were done using fungal strains from the collection of the Department of Pathogen Genetics and Plant Resistance, Institute of Plant Genetics, Polish Academy of Sciences. The fungi were isolated from the leaves of oilseed rape plants, single-spored and classified as Plenodomus lingam (formerly Leptosphaeria maculans) and P. biglobosus subclade 'brassicae' (formerly L. biglobosa subclade 'brassicae') based on their morphology and molecular characteristics, as described before [53,70]. There were three isolates of P. lingam (PL1, PL2, and PL3) and three isolates of P. biglobosus subclade 'brassicae' (PB1, PB2, and PB3) studied.

Filamentous Fungi (FF) Plates Assay
The metabolic capacity and profile of six Plenodomus isolates, including three isolates of P. lingam and three isolates of P. biglobosus subclade 'brassicae', was measured using filamentous fungi (FF) microplates (Biolog, Inc., Hayward, CA, USA) containing 95 different carbon sources and tetrazolium dye. The fungi were cultured on potato dextrose agar (PDA) for 6 weeks at 20 • C and then fungal spores were used as inoculum of microplates. The inoculation procedure was performed according to manufacturer protocol with modifications described by Frąc [71] and Oszust et al. [72] in triplicate using three separate plates for each fungal isolate. In brief, after homogenization of fungal spores in distilled sterile water with Pulsifier apparatus, the obtained fungal suspension in inoculating fluid (FF-IF, Biolog) containing Phytagel, Tween 40 and water was adjusted to 75% of transmittance using turbidimeter (Biolog, Inc., Hayward, CA, USA).
Then, 100 µL of the above-mentioned fungal spores suspension were added into each well of FF microplates and the plates were incubated at 27 • C in darkness within 240 h. The measurements of absorbance at wavelength 490 nm (substrate utilization) and 750 nm (fungal growth) were performed every 24-h using a microplate reader (Biolog Inc., Hayward, CA, USA).

Metabolic Capacity, Fungal Growth and Group of Substrates Use
Based on measurement at 490 nm and 750 nm, average well color development (AWCD) and average well density development (AWDD) indices were calculated, respectively [71,73]. The substrate richness indices presenting the number of different substrates utilized by the strain (490 nm) or used by fungi to grow (750 nm) were calculated as all positive readings with the threshold ≥0. 25. The response of each tested fungal strain to individual 95 carbon substrates was assessed as a level of consumption of different substrates and growth intensity on substrates. The percentage of the use of six main groups of carbon sources (amines and amides, amino acids, carbohydrates, carboxylic acids, polymers, miscellaneous), as well as the fungal abilities to grow on them, were calculated to present the response of tested fungal pathogens to substrates. Moreover, the level of metabolic capacity and fungal growth intensity on those groups of carbon sources were calculated. To analyze deeper the carbon sources guild utilization, fifteen groups of substrates were evaluated according to Atanasova and Druzhinina [74], based on their chemical structure and properties. The following guilds of substrates were tested: heptoses, hexoses, pentoses, sugar acids, hexosamines, polyols, polysaccharides, oligosaccharides, glucosides, peptides, L-amino acids, biogenic and heterocyclic amines, TCA-cycle intermediates, aliphatic organic acids, and others. The ratio of AWCD and AWDD of substrate group [75,76] and the particular carbon sources for tested fungi was calculated to indicate the specific respiration rate for the mean values of each substrate group and shows catabolic capacity compared with fungal biomass production. The higher ratio indicates the higher stressful metabolic situation, showing lower biomass production and higher respiration rates [77,78].

Statistical Analysis
Data analysis was performed with the STATISTICA 13.0 (StatSoft Inc., Tulsa, OK, USA) software package. One-way analysis of variance ANOVA (with confidence interval 95%) was performed to compare the growth of selected strains on individual carbon sources and the level of substrate utilization, expressed as AWDD, AWCD, and R indices. Then, the indices were assessed by two-way ANOVA analysis regarding the effect of the incubation time and tested fungal isolates. ANOVA was followed by a post hoc analysis using Tukey's HSD (Honestly Significant Difference) test. The summed data matrices also were evaluated following multidimensional scaling to detect additional relationships between variables. Cluster analysis was performed to detect groups in the data set. To illustrate the results, the similarities of the carbon utilization patterns between the strains were presented using heat maps graphs and the percentage of total carbon source utilization. As a function of the carbon utilization a dendrogram calculated with the Ward method [79] and Sneath dissimilarity criterion [80] was performed to indicate the dissimilarity of fungal groups based on their response to substrates tested.